RESUMO
BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.
Assuntos
Artefatos , Genoma Humano , Humanos , Biblioteca Gênica , Análise de Sequência de DNA/métodos , DNA de Neoplasias , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
H subunit of V-ATPase (ATP6V1H) is specifically expressed in osteoclasts and its deficiency lead to osteoporosis. Our group previously found four intronic SNPs of ATP6V1H related to reduced bone mineral density, but the mechanisms was not clear. In this study, we found that the above four SNPs were located at lncRNA lnc-TCEA1-3 by using bioinformatics analysis. We further detected the function of lnc-TCEA1-3 on regulating ATP6V1H and osteoclast function using Atp6v1h knockout mice, lentivirus transfection and qPCR analysis. Over expression of lnc-TCEA1-3 up regulated the expression of ATP6V1H in HEK293 cells, HOS cells and primarily cultured osteoclasts, and increased the number of primarily cultured osteoclasts. In addition, over expression of lnc-TCEA1-3 exerted distinct effect on two transcripts of ATP6V1H in HEK293, HOS and osteoclasts. This study will facilitate the in-depth analysis of the effects of ATP6V1H on bone diseases, and discover new therapeutic strategies.
Assuntos
Osteoporose , RNA Longo não Codificante , ATPases Vacuolares Próton-Translocadoras , Animais , Camundongos , Humanos , Osteoclastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células HEK293 , Osteoporose/genética , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Updated and expert-quality knowledge bases are fundamental to biomedical research. A knowledge base established with human participation and subject to multiple inspections is needed to support clinical decision making, especially in the growing field of precision oncology. The number of original publications in this field has risen dramatically with the advances in technology and the evolution of in-depth research. Consequently, the issue of how to gather and mine these articles accurately and efficiently now requires close consideration. In this study, we present OncoPubMiner (https://oncopubminer.chosenmedinfo.com), a free and powerful system that combines text mining, data structure customisation, publication search with online reading and project-centred and team-based data collection to form a one-stop 'keyword in-knowledge out' oncology publication mining platform. The platform was constructed by integrating all open-access abstracts from PubMed and full-text articles from PubMed Central, and it is updated daily. OncoPubMiner makes obtaining precision oncology knowledge from scientific articles straightforward and will assist researchers in efficiently developing structured knowledge base systems and bring us closer to achieving precision oncology goals.
Assuntos
Neoplasias , Mineração de Dados , Humanos , Oncologia , Medicina de Precisão , PubMed , PublicaçõesRESUMO
Astragaloside IV (AST) has been confirmed to have antiasthmatic effects. However, the underline mechanism is unclear. The study aimed to explore the treatment mechanism of AST based on autophagy of memory T cells. AST treatment significantly decreased the number of T effector cells in asthma mice blood and the nude mice that received AST-treated TCMs had relieved inflammation compared with the untreated group; meanwhile, we found that AST significantly decreased the autophagy level and inhibited OX40/OX40L signal pathway of lymphocytes. The results highlighted that AST regulated autophagy to inhibit differentiation of effector T-cell phenotype.
Assuntos
Asma , Autofagia , Inflamação , Saponinas , Linfócitos T , Triterpenos , Animais , Saponinas/farmacologia , Asma/tratamento farmacológico , Triterpenos/farmacologia , Triterpenos/química , Camundongos , Autofagia/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Inflamação/tratamento farmacológico , Camundongos Nus , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
The microgravity conditions in outer space are widely acknowledged to induce significant bone loss. Recent studies have implicated the close relationship between Atp6v1h gene and bone loss. Despite this, the role of Atp6v1h in bone remodeling and its molecular mechanisms in microgravity have not been fully elucidated. To address this, we used a mouse tail suspension model to simulate microgravity. We categorized both wild-type and Atp6v1h knockout (Atp6v1h+/-) mice into two groups: regular feeding and tail-suspension feeding, ensuring uniform feeding conditions across all cohorts. Analysis via micro-CT scanning, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase assays indicated that wild-type mice underwent bone loss under simulated microgravity. Atp6v1h+/- mice exhibited bone loss due to Atp6v1h deficiency but did not present aggravated bone loss under the same simulated microgravity. Transcriptomic sequencing revealed the upregulation of genes, such as Fos, Src, Jun, and various integrin subunits in the context of simulated microgravity and Atp6v1h knockout. Real-time quantitative polymerase chain reaction (RT-qPCR) further validated the modulation of downstream osteoclast-related genes in response to interactions with ATP6V1H overexpression cell lines. Co-immunoprecipitation indicated potential interactions between ATP6V1H and integrin beta 1, beta 3, beta 5, alpha 2b, and alpha 5. Our results indicate that Atp6v1h level influences bone loss in simulated microgravity by modulating the Fos-Jun-Src-Integrin pathway, which, in turn, affects osteoclast activity and bone resorption, with implications for osteoporosis. Therefore, modulating Atp6v1h expression could mitigate bone loss in microgravity conditions. This study elucidates the molecular mechanism of Atp6v1h's role in osteoporosis and positions it as a potential therapeutic target against environmental bone loss. These findings open new possibilities for the treatment of multifactorial osteoporosis.
Assuntos
Doenças Ósseas Metabólicas , Osteoporose , ATPases Vacuolares Próton-Translocadoras , Ausência de Peso , Animais , Camundongos , Modelos Animais de Doenças , Integrinas , Osteoporose/genética , Ausência de Peso/efeitos adversos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Next-generation sequencing (NGS) technology has revolutionised human cancer research, particularly via detection of genomic variants with its ultra-high-throughput sequencing and increasing affordability. However, the inundation of rich cancer genomics data has resulted in significant challenges in its exploration and translation into biological insights. One of the difficulties in cancer genome sequencing is software selection. Currently, multiple tools are widely used to process NGS data in four stages: raw sequence data pre-processing and quality control (QC), sequence alignment, variant calling and annotation and visualisation. However, the differences between these NGS tools, including their installation, merits, drawbacks and application, have not been fully appreciated. Therefore, a systematic review of the functionality and performance of NGS tools is required to provide cancer researchers with guidance on software and strategy selection. Another challenge is the multidimensional QC of sequencing data because QC can not only report varied sequence data characteristics but also reveal deviations in diverse features and is essential for a meaningful and successful study. However, monitoring of QC metrics in specific steps including alignment and variant calling is neglected in certain pipelines such as the 'Best Practices Workflows' in GATK. In this review, we investigated the most widely used software for the fundamental analysis and QC of cancer genome sequencing data and provided instructions for selecting the most appropriate software and pipelines to ensure precise and efficient conclusions. We further discussed the prospects and new research directions for cancer genomics.
Assuntos
Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Controle de Qualidade , Biologia Computacional/métodos , Humanos , Anotação de Sequência Molecular , SoftwareRESUMO
Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3-ITD) constitutes an independent indicator of poor prognosis in acute myeloid leukaemia (AML). AML with FLT3-ITD usually presents with poor treatment outcomes, high recurrence rate and short overall survival. Currently, polymerase chain reaction and capillary electrophoresis are widely adopted for the clinical detection of FLT3-ITD, whereas the length and mutation frequency of ITD are evaluated using fragment analysis. With the development of sequencing technology and the high incidence of FLT3-ITD mutations, a multitude of bioinformatics tools and pipelines have been developed to detect FLT3-ITD using next-generation sequencing data. However, systematic comparison and evaluation of the methods or software have not been performed. In this study, we provided a comprehensive review of the principles, functionality and limitations of the existing methods for detecting FLT3-ITD. We further compared the qualitative and quantitative detection capabilities of six representative tools using simulated and biological data. Our results will provide practical guidance for researchers and clinicians to select the appropriate FLT3-ITD detection tools and highlight the direction of future developments in this field. Availability: A Docker image with several programs pre-installed is available at https://github.com/niu-lab/docker-flt3-itd to facilitate the application of FLT3-ITD detection tools.
Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Duplicação Gênica , Leucemia Mieloide/genética , Sequências de Repetição em Tandem/genética , Tirosina Quinase 3 Semelhante a fms/genética , Doença Aguda , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide/diagnóstico , MutaçãoRESUMO
MOTIVATION: Microsatellite instability (MSI) is a promising biomarker for cancer prognosis and chemosensitivity. Techniques are rapidly evolving for the detection of MSI from tumor-normal paired or tumor-only sequencing data. However, tumor tissues are often insufficient, unavailable, or otherwise difficult to procure. Increasing clinical evidence indicates the enormous potential of plasma circulating cell-free DNA (cfNDA) technology as a noninvasive MSI detection approach. RESULTS: We developed MSIsensor-ct, a bioinformatics tool based on a machine learning protocol, dedicated to detecting MSI status using cfDNA sequencing data with a potential stable MSIscore threshold of 20%. Evaluation of MSIsensor-ct on independent testing datasets with various levels of circulating tumor DNA (ctDNA) and sequencing depth showed 100% accuracy within the limit of detection (LOD) of 0.05% ctDNA content. MSIsensor-ct requires only BAM files as input, rendering it user-friendly and readily integrated into next generation sequencing (NGS) analysis pipelines. AVAILABILITY: MSIsensor-ct is freely available at https://github.com/niu-lab/MSIsensor-ct. SUPPLEMENTARY INFORMATION: Supplementary data are available at Briefings in Bioinformatics online.
Assuntos
DNA Tumoral Circulante/genética , Aprendizado de Máquina , Instabilidade de Microssatélites , Neoplasias/genética , Software , DNA Tumoral Circulante/sangue , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Limite de Detecção , Repetições de Microssatélites , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/patologia , Análise de Sequência de DNARESUMO
Tooth eruption is an important and unique biological process during craniofacial development. Both the genetic and environmental factors can interfere with this process. Here we aimed to find the failure pattern of tooth eruption among five genetic diseases. Both systematic review and meta-analysis were used to identify the genotype-phenotype associations of unerupted teeth. The meta-analysis was based on the characteristics of abnormal tooth eruption in 223 patients with the mutations in PTH1R, RUNX2, COL1A1/2, CLCN7, and FAM20A respectively. We found all the patients presented selective failure of tooth eruption (SFTE). Primary failure of eruption patients with PTH1R mutations showed primary or isolated SFTE1 in the first and second molars (59.3% and 52% respectively). RUNX2 related cleidocranial dysplasia usually had SFTE2 in canines and premolars, while COL1A1/2 related osteogenesis imperfecta mostly caused SFTE3 in the maxillary second molars (22.9%). In CLCN7 related osteopetrosis, the second molars and mandibular first molars were the most affected. While FAM20A related enamel renal syndrome most caused SFTE5 in the second molars (86.2%) and maxillary canines. In conclusion, the SFTE was the common characteristics of most genetic diseases with abnormal isolated or syndromic tooth eruption. The selective pattern of unerupted teeth was gene-dependent. Here we recommend SFTE to classify those genetic unerupted teeth and guide for precise molecular diagnosis and treatment.
Assuntos
Anormalidades Dentárias , Dente não Erupcionado , Humanos , Erupção Dentária/genética , Dente não Erupcionado/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fenótipo , Genótipo , Canais de Cloreto/genéticaRESUMO
Amelogenesis imperfecta (AI) is one of the typical dental genetic diseases in human. It can occur isolatedly or as part of a syndrome. Previous reports have mainly clarified the types and mechanisms of nonsyndromic AI. This review aimed to compare the phenotypic differences among the hereditary enamel defects with or without syndromes and their underlying pathogenic genes. We searched the articles in PubMed with different strategies or keywords including but not limited to amelogenesis imperfecta, enamel defects, hypoplastic/hypomaturation/hypocalcified, syndrome, or specific syndrome name. The articles with detailed clinical information about the enamel and other phenotypes and clear genetic background were used for the analysis. We totally summarized and compared enamel phenotypes of 18 nonsyndromic AI with 17 causative genes and 19 syndromic AI with 26 causative genes. According to the clinical features, radiographic or ultrastructural changes in enamel, the enamel defects were basically divided into hypoplastic and hypomineralized (hypomaturated and hypocalcified) and presented a higher heterogeneity which were closely related to the involved pathogenic genes, types of mutation, hereditary pattern, X chromosome inactivation, incomplete penetrance, and other mechanisms.The gene-specific enamel phenotypes could be an important indicator for diagnosing nonsyndromic and syndromic AI.
Assuntos
Amelogênese Imperfeita , Hipoplasia do Esmalte Dentário , Proteínas do Esmalte Dentário , Humanos , Amelogênese Imperfeita/genética , Amelogênese Imperfeita/patologia , Esmalte Dentário/química , Proteínas do Esmalte Dentário/genética , FenótipoRESUMO
OBJECTIVE: This review aimed to summarize recent progress on syndromic dentin defects, promoting a better understanding of systemic diseases with dentin malformations, the molecules involved, and related mechanisms. SUBJECTS AND METHODS: References on genetic diseases with dentin malformations were obtained from various sources, including PubMed, OMIM, NCBI, and other websites. The clinical phenotypes and genetic backgrounds of these diseases were then summarized, analyzed, and compared. RESULTS: Over 10 systemic diseases, including osteogenesis imperfecta, hypophosphatemic rickets, vitamin D-dependent rickets, familial tumoral calcinosis, Ehlers-Danlos syndrome, Schimke immuno-osseous dysplasia, hypophosphatasia, Elsahy-Waters syndrome, Singleton-Merten syndrome, odontochondrodysplasia, and microcephalic osteodysplastic primordial dwarfism type II were examined. Most of these are bone disorders, and their pathogenic genes may regulate both dentin and bone development, involving extracellular matrix, cell differentiation, and metabolism of calcium, phosphorus, and vitamin D. The phenotypes of these syndromic dentin defects various with the involved genes, part of them are similar to dentinogenesis imperfecta or dentin dysplasia, while others only present one or two types of dentin abnormalities such as discoloration, irregular enlarged or obliterated pulp and canal, or root malformation. CONCLUSION: Some specific dentin defects associated with systemic diseases may serve as important phenotypes for dentists to diagnose. Furthermore, mechanistic studies on syndromic dentin defects may provide valuable insights into isolated dentin defects and general dentin development or mineralization.
Assuntos
Dentinogênese Imperfeita , Odontodisplasia , Osteogênese Imperfeita , Humanos , Dentinogênese Imperfeita/genética , Odontodisplasia/patologia , Osteogênese Imperfeita/patologia , Dentina , Vitamina DRESUMO
OBJECTIVE: Muscle segment homeobox gene 1 (MSX1) is widely expressed in craniofacial development and tooth formation. The aim of this study was to report a novel MSX1 mutation in a Chinese family with selective tooth agenesis and abnormal median maxillary labial frenum (MMLF). MATERIALS AND METHODS: Mutation analysis was carried out by whole exome sequencing. The pMD18-T vector was used to verify the mutations. PubMed and Human Gene Mutation Database were searched to analyze the relationship between the mutations in MSX1 and related phenotypes. RESULTS: A novel heterozygous mutation (c.75delG) in MSX1 was detected in the proband and her mother. They presented as oligodontia and lower attached hypertrophy median maxillary labial frenum. 60 MSX1 mutations from 39 reports did not declare malformed MMLF except our cases. Meanwhile, we found that the types and sites of MSX1 mutations may affect the selectivity of tooth agenesis and orofacial cleft. CONCLUSION: This study suggests malformed MMLF as a new phenotype of MSX1 mutation and a specific relationship between MSX1 genotype and phenotype.
Assuntos
Anodontia , Fenda Labial , Fissura Palatina , Humanos , Feminino , Estudos Retrospectivos , Freio Labial , Fenda Labial/genética , Linhagem , Anodontia/genética , Mutação , Fator de Transcrição MSX1/genéticaRESUMO
BACKGROUND: Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice. METHODS: A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples. RESULTS: The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples. CONCLUSIONS: The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.
Assuntos
Neoplasias , Ácidos Nucleicos , Humanos , Neoplasias/genética , RNA/genética , Sondas RNA , Medicina de Precisão , DNA , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Osteopetrosis is a group of genetic bone disorders characterized by increased bone density and defective bone resorption. Osteopetrosis presents a series of clinical manifestations, including craniofacial deformities and dental problems. However, few previous reports have focused on the features of craniofacial and dental problems in osteopetrosis. In this review, we go through the clinical features, types, and related pathogenic genes of osteopetrosis. Then we summarize and describe the characteristics of craniofacial and dental abnormalities in osteopetrosis that have been published in PubMed from 1965 to the present. We found that all 13 types of osteopetrosis have craniomaxillofacial and dental phenotypes. The main pathogenic genes, such as chloride channel 7 gene (CLCN7), T cell immune regulator 1 (TCIRG1), osteopetrosis-associated transmembrane protein 1 (OSTM1), pleckstrin homology domain-containing protein family member 1 (PLEKHM1), and carbonic anhydrase II (CA2), and their molecular mechanisms involved in craniofacial and dental phenotypes, are discussed. We conclude that the telltale craniofacial and dental abnormalities are important for dentists and other clinicians in the diagnosis of osteopetrosis and other genetic bone diseases.
Assuntos
Reabsorção Óssea , Osteopetrose , ATPases Vacuolares Próton-Translocadoras , Humanos , Osteopetrose/genética , Osteopetrose/patologia , Osso e Ossos/metabolismo , Fenótipo , Canais de Cloreto/metabolismo , Mutação , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The solute carrier family 4 (SLC4) is an important protein responsible for the transport of various ions across the cell membrane and mediating diverse physiological functions, such as the ion transporting function, protein-to-protein interactions, and molecular transduction. The deficiencies in SLC4 molecules may cause multisystem disease involving, particularly, the respiratory system, digestive, urinary, endocrine, hematopoietic, and central nervous systems. Currently, there are no effective strategies to treat these diseases. SLC4 proteins are also found to contribute to tumorigenesis and development, and some of them are regarded as therapeutic targets in quite a few clinical trials. This indicates that SLC4 proteins have potential clinical prospects. In view of their functional characteristics, there is a critical need to review the specific functions of bicarbonate transporters, their related diseases, and the involved pathological mechanisms. We summarize the diseases caused by the mutations in SLC4 family genes and briefly introduce the clinical manifestations of these diseases as well as the current treatment strategies. Additionally, we illustrate their roles in terms of the physiology and pathogenesis that has been currently researched, which might be the future therapeutic and diagnostic targets of diseases and a new direction for drug research and development.
Assuntos
Medicina de Precisão , Humanos , Transporte de Íons/fisiologia , MutaçãoRESUMO
The solute carrier family 4 (SLC4) includes 10 members (SLC4A1-5, SLC4A7-11), which are expressed in multiple tissues in the human body. The SLC4 family members differ in their substrate dependence, charge transport stoichiometry and tissue expression. Their common function is responsible for the transmembrane exchange of multiple ions, which is involved in many important physiological processes, such as erythrocyte CO2 transport and the regulation of cell volume and intracellular pH. In recent years, many studies have focused on the role of SLC4 family members in the occurrence of human diseases. When SLC4 family members have gene mutations, a series of functional disorders will occur in the body, leading to the occurrence of some diseases. This review summarizes the recent progress about the structures, functions and disease correlation of SLC4 members, in order to provide clues for the prevention and treatment of related human diseases.
Assuntos
Mutação , Proteínas SLC4A , Humanos , Proteínas SLC4A/genética , Proteínas SLC4A/fisiologiaRESUMO
Vacuolar H+-ATPase (V-ATPase) is a ubiquitous proton pump that mediates the proton transmembrane transportation in various cells. Previously, H subunit of V-ATPase (ATP6V1H) was found to be related with insulin secretion and diabetes. However, the mechanism by which ATP6V1H regulates insulin secretion and glucose metabolism remains unclear. Herein, we established a high-fat-diet (HFD) fed model with Atp6v1h+/- mice and detected the expression and secretion of insulin and some biochemical indices of glucose metabolism, in order to explore the related mechanisms in ß-cells. Transcriptome sequencing, qPCR and western blot analysis showed that ATP6V1H deficiency worsened fatty acid-induced glucose tolerance impairment by augmenting endoplasmic reticulum stress in ß-cells, and alternative splicing of ATP6V1H might be involved in this process. These results indicated that ATP6V1H deficiency increased the susceptibility to T2DM.
Assuntos
Metabolismo dos Carboidratos/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina , Masculino , CamundongosRESUMO
BACKGROUND: African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects. RESULTS: The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98). CONCLUSIONS: The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Anticorpos de Domínio Único , Doenças dos Suínos , Vírus da Febre Suína Africana/genética , Animais , Camelus , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Peroxidase do Rábano Silvestre , Indicadores e Reagentes , Reprodutibilidade dos Testes , SuínosRESUMO
MOTIVATION: HotSpot3D is a widely used software for identifying mutation hotspots on the 3D structures of proteins. To further assist users, we developed a new HotSpot3D web server to make this software more versatile, convenient and interactive. RESULTS: The HotSpot3D web server performs data pre-processing, clustering, visualization and log-viewing on one stop. Users can interactively explore each cluster and easily re-visualize the mutational clusters within browsers. We also provide a database that allows users to search and visualize proximal mutations from 33 cancers in the Cancer Genome Atlas. AVAILABILITY AND IMPLEMENTATION: http://niulab.scgrid.cn/HotSpot3D/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas , Software , Computadores , Bases de Dados Factuais , Internet , Mutação , Proteínas/genéticaRESUMO
The role of circular RNA (circRNA) pappalysin 1 (circ-PAPPA; hsa_circ_0088233) in prostate cancer (PCa) cells was explored in the current study. Circ-PAPPA abundance was markedly enhanced in PCa. Circ-PAPPA interference restrained cell viability, proliferation, motility and glycolysis while elevated the apoptosis rate of PCa cells. Circ-PAPPA negatively regulated microRNA-515-5p (miR-515-5p) abundance. MiR-515-5p silencing largely diminished circ-PAPPA knockdown-mediated effects in PCa cells. MiR-515-5p directly bound to FKBP prolyl isomerase 1A (FKBP1A). MiR-515-5p overexpression-mediated impacts were partly counteracted by FKBP1A overexpression. Circ-PAPPA silencing reduced FKBP1A protein level partly by elevating miR-515-5p expression. Circ-PAPPA knockdown significantly restrained the tumour growth in vivo. Circ-PAPPA elevated the malignant phenotypes of PCa cells by sequestering miR-515-5p to induce the expression of FKBP1A.